The vertebrate immune response to infection begins with the recognition by the innate immune system of conserved molecular signatures of pathogens, known as PAMPs (Pathogen Associated Molecular Patterns), provoking an immediate and often massive inflammatory response. The innate response holds the pathogen in check, but also plays a crucial role in the generation of acquired immunity. The recognition of PAMPs by the innate system is mediated by a number of receptors, the most important of which are the TLRs. Unlike the antigen receptors of acquired immunity, the TLRs are encoded by a limited number of germline genes, ten in humans; however, in spite of their small numbers, the TLRs recognize a remarkably wide variety of PAMPs including glycolipids, proteins, and nucleic acids. We have been investigating four aspects of TLR structure and function.I. How the TLRs recognize such a wide array of PAMPs is a main interest of my laboratory. In collaboration with Dr. David Davies, we have recently succeeded in expressing, crystallizing, and determining the molecular structure of the TLR3 extracellular domain (ECD). The structure is quite elegant; it consists of a solenoid of 23 turns, bent into a horseshoe shape, with a large b-sheet on the concave surface. Our immediate goal is to determine at the molecular level, how the TLR3 horseshoe specifically recognizes its ligand, dsRNA, and how recognition leads to activation. In addition to X-ray analysis, we are currently studying the binding of various dsRNA ligands to TLR3-ECD protein in solution, and we are in the process of introducing mutations to determine which residues are essential for activity. Looking past TLR3, we plan to express and examine ECDs from other TLR paralogs, to see how they differ in structure and ligand binding function from TLR3. II. We are studying the interaction of TLR4 with its ligand, lipopolysaccharide (LPS), a causative agent of Gram negative sepsis. Four proteins, LBP, CD14, TLR4, and MD-2 are known to be involved in the mammalian response to LPS, but the essential signaling receptor for LPS consists of a complex containing only MD-2 and TLR4. MD-2 was originally discovered as a small glycoprotein bound to the ECD of TLR4 on the cell surface, and it was proposed that the interaction of TLR4 with MD-2 occurred intracellularly. However, we found that in addition to binding to TLR4 in the ER, MD-2 was also secreted into the medium, and that the secreted form (sMD-2) had several interesting properties. sMD-2 is able to confer LPS responsiveness to cells, such as epithelial cells and HEK293 transfectants, that express TLR4 but not MD-2. In solution, sMD-2 rapidly loses activity at physiological temperature, but is stabilized by LPS. We showed that a stable complex between LPS and MD-2, and not LPS itself, is the actual activating ligand of TLR4, and that CD14 is required only to transfer LPS to MD-2. To date we have demonstrated that MD-2/LPS binds and activates free TLR4, but we are also in the process of determining whether it can activate preformed TLR4/MD-2 complexes, and if so what is the mechanism. These studies will be especially important in understanding the sensitivity and control of the response to LPS in vivo. III. Nucleic acid PAMPs such as dsRNA, ssRNA, and CpG DNA, ligands for TLRs 3, 7, 8, and 9, are normally sequestered within microorganisms and become available to interact with TLRs only after the pathogen is endocytosed and lysed intracellularly. By contrast, TLRs 1, 2, 4, 5, 6, and 10 interact with PAMPs that are normally present in the medium, and these TLRs are, as expected, located on the cell surface. Therefore, correct cellular localization is essential for TLR function. We are currently studying the localization of TLR9, and motifs within the TLR9 molecule that mediate this localization.